Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

Influence of microgravity on root-cap regeneration and the structure of columella cells in Zea mays

We launched imbibed seeds and seedlings of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on 1) root-cap regeneration, and 2) the distribution of amyloplasts and endoplasmic reticulum (ER) in the putative statocytes (i.e., columella cells) of roots. Decapped roots grown on Earth completely regenerated their caps within 4.8 days after decapping, while those grown in microgravity did not regenerate caps. In Earth-grown seedlings, the ER was localized primarily along the periphery of columella cells, and amyloplasts sedimented in response to gravity to the lower sides of the cells. Seeds germinated on Earth and subsequently launched into outer space had a distribution of ER in columella cells similar to that of Earth-grown controls, but amyloplasts were distributed throughout the cells. Seeds germinated in outer space were characterized by the presence of spherical and ellipsoidal masses of ER and randomly distributed amyloplasts in their columella cells. These results indicate that 1) gravity is necessary for regeneration of the root cap, 2) columella cells can maintain their characteristic distribution of ER in microgravity only if they are exposed previously to gravity, and 3) gravity is necessary to distribute the ER in columella cells of this cultivar of Z. mays.     

Gravitropism in the starch excess mutant of Arabidopsis thaliana

Amyloplasts are hypothesized to play a key role in the cellular mechanisms of gravity perception in plants. While previous studies have examined the effects of starch deficiency on gravitropic sensitivity, in this paper, we report on gravitropism in plants with a greater amount of starch relative to the normal wild type. Thus, we have studied the sex1 (starch excess) mutant of Arabidopsis thaliana, which accumulates extra starch because it is defective in a protein involved in the regulation of starch mobilization. Compared to the wild type (WT), sex1 seedlings contained excess starch in cotyledons, hypocotyls, the root-hypocotyl transition zone, the body of the root, root hairs, and in peripheral rootcap cells. Sedimented amyloplasts were found in both the WT and in sex1 in the rootcap columella and in the endodermis of stems, hypocotyls, and petioles. In roots, the starch content and amyloplast sedimentation in central columella cells and the gravitropic sensitivity were comparable in sex1 and the WT. However, in hypocotyls, the sex1 mutant was much more sensitive to gravity during light-grown conditions compared to the WT. This difference was correlated to a major difference in size of plastids in gravity-perceiving endodermal cells between the two genotypes (i.e., sex1 amyloplasts were twice as big). These results are consistent with the hypothesis that only very large changes in starch content relative to the WT affect gravitropic sensitivity, thus indicating that wild-type sensing is not saturated.     

Plastid Distribution in Columella Cells of a Starchless Arabidopsis Mutant Grown in Microgravity

Wild-type and starchless Arabidopsis thaliana mutant seedlings(TC7) were grown and fixed in the microgravity environment ofa U.S. Space Shuttle spaceflight. Computer image analysis oflongitudinal sections from columella cells suggest a differentplastid positioning mechanism for mutant and wild-type in theabsence of gravity. (Received September 24, 1996; Accepted January 21, 1997)     

Statolith Sedimentation Kinetics and Force Transduction to the Cortical Endoplasmic Reticulum in Gravity-Sensing Arabidopsis Columella Cells

The starch statolith hypothesis of gravity sensing in plants postulates that the sedimentation of statoliths in specialized statocytes (columella cells) provides the means for converting the gravitational potential energy into a biochemical signal. We have analyzed the sedimentation kinetics of statoliths in the central S2 columella cells of Arabidopsis thaliana. The statoliths can form compact aggregates with gap sizes between statoliths approaching <30 nm. Significant intra-aggregate sliding motions of individual statoliths suggest a contribution of hydrodynamic forces to the motion of statoliths. The reorientation of the columella cells accelerates the statoliths toward the central cytoplasm within <1 s of reorientation. During the subsequent sedimentation phase, the statoliths tend to move at a distance to the cortical endoplasmic reticulum (ER) boundary and interact only transiently with the ER. Statoliths moved by laser tweezers against the ER boundary experience an elastic lift force upon release from the optical trap. High-resolution electron tomography analysis of statolith-to-ER contact sites indicate that the weight of statoliths is sufficient to locally deform the ER membranes that can potentially activate mechanosensitive ion channels. We suggest that in root columella cells, the transduction of the kinetic energy of sedimenting statoliths into a biochemical signal involves a combination of statolith-driven motion of the cytosol, statolith-induced deformation of the ER membranes, and a rapid release of kinetic energy from the ER during reorientation to activate mechanosensitive sites within the central columella cells.     

A morphometric analysis of the redistribution of organelles in columella cells in primary roots of normal seedlings and agravitropic mutants of Hordeum vulgare

The redistribution of organelles in columella cells of horizontally-oriented roots of Hordeum vulgare was quantified in order to determine what structural changes in graviperceptive (i.e., columella) cells are associated with the onset of the root gravicurvature. The sedimentation of amyloplasts is the only major change in cellular structure that correlates positively with the onset of root gravicurvature, which begins within 15 min after re-orientation. There is no consistent contact between sedimented amyloplasts and any other organelles. Nuclei are restricted to the proximal ends of columella cells in vertically-oriented roots, and remain there throughout gravicurvature after roots are oriented horizontally. Root gravicurvature does not involve significant changes in (1) the volume of columella cells, (2) the relative or absolute volumes of organelles in columella cells, or (3) the distribution of endoplasmic reticulum (ER). The size, number and sedimentation rates of amyloplasts in columella cells of non-graviresponsive roots of mutant seedlings are not significantly different from those of graviresponsive roots of normal seedlings. Similarly, there is no significant difference in (1) cellular volume, (2) distribution or surface area of ER, (3) patterns or rates of organelle redistribution in horizontally-oriented roots, (4) relative or absolute volumes of organelles in columella cells of graviresponsive and non-graviresponsive roots. These results suggest that the lack of graviresponsiveness by roots of mutant seedlings is probably not due to either (1) structural differences in columella cells, or (2) differences in patterns or rates of organelle redistribution as compared to that characteristic of graviresponsive roots. Thus, the basis of non-graviresponsiveness in this mutant is probably different from other agravitropic mutants so far studied.     

Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover ( Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.     

Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells

The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.     

Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids

The sites of gravity perception are columella cells in roots and endodermal cells in hypocotyls and inflorescence stems. Since plastids are likely to play a role in graviperception, we investigated gravitropism in plastid mutants of Arabidopsis . Previous studies have shown that the arc 6 and arc 12 ( a ccumulation and r eplication of c hloroplasts) mutants have an average of two large plastids per leaf mesophyll cell. In this study, we found that these arc mutants have altered plastid morphology throughout the entire plant body, including the cells involved in gravity perception. There were no major differences in total starch content per cell in endodermal and columella cells of the wild-type (WT) compared to arc 6 and arc 12 as assayed by iodine staining. Thus, the total mass of plastids per cell in arc 6 and arc 12 is similar to their respective WT strains. Results from time course of curvature studies demonstrated that the plastid mutation affected gravitropism only of inflorescence stems and hypocotyls, but not roots. Thus, roots appear to have different mechanisms of gravitropism compared to stems and hypocotyls. Time course of curvature studies with light-grown seedlings were performed in the presence of latrunculin B (Lat-B), an actin-depolymerizing drug. Lat-B promoted gravitropic curvature in hypocotyls of both the WT and arc 6 but had little or no effect on gravitropism in roots of both strains. These results suggest that F-actin is not required for hypocotyl gravitropism.     

Ultrastructure and movements of cell organelles in the root cap of agravitropic mutants and normal seedlings of Arabidopsis thaliana

The root anatomy and ultrastructure of the agravitropic Arabidopsis thaliana L. mutants Dwf and aux-1 were compared with the gravitropic mutant aux-2 and the wild type (WT) in an attempt to find an explanation for the lack of response to gravity. No differences were found in the organization of the root cap. The central part of the cap (columella) contains 5 storeys of developing, functioning and degenerating statocytes. Their ultrastructure is very similar in all four types of plant. Particular attention was paid to the distribution of rough endoplasmie reticulum (ER). Both in the WT and the mutants the ER is concentrated in the distal part at the "floor" of the cell.
Light micrographs were used to compare the sedimentation rates of movable cell structures in normal and agravitropic root statocytes. A longitudinal movement of amyloplasts and nuclei was observed when the roots were inverted. In WT and aux-2 the rates were on average 6.3 μm h⁻¹ (amyloplasts) and 2.1 μm h⁻¹ (nucleus). In aux-1 the sedimentation rates were significantly lower: 2.4 and 0.6 μm h⁻¹, respectively. Based on magnified electron micrographs of normal and inverted statocytes a morphometrical analysis of the distribution and redistribution of amyloplasts, nuclei, mitochondria, vacuoles and ER was made. The only significant difference was found in the redistribution of amyloplasts between aux-1 and the gravitropical normal types.     

Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity

Despite the extensive study of plant gravitropism, there have been few experiments which have utilized hypergravity as a tool to investigate gravisensitivity in flowering plants. Previous studies have shown that starch-deficient mutants of Arabidopsis are less sensitive to gravity compared to the wild-type (WT). In this report, the question addressed was whether hypergravity could restore the sensitivity of starch-deficient mutants of Arabidopsis. The strains examined include a WT, a starchless mutant and a reduced-starch mutant. Vertical orientation studies with dark-grown seedlings indicate that increased centrifugal acceleration improves orientation relative to the acceleration vector for all strains, even the WT. For starchless roots, growth of seedlings under constant 5 g acceleration was required to restore orientation to the level of the WT at 1 g. In contrast, approximately 10 g was required to restore the orientation of the starchless mutant hypocotyls to a WT level at 1 g. Examination of plastid position in root cap columella cells of the starchless mutant revealed that the restoration of gravitropic sensitivity was correlated with the sedimentation of plastids toward the distal cell wall. Even in WT plants, hypergravity caused greater sedimentation of plastids and improved gravitropic capability. Collectively, these experiments support the hypothesis of a statolith-based system of gravity perception in plants. As far as is known, this is the first report to use hypergravity to study the mechanisms of gravitropism in Arabidopsis.     

Amyloplast sedimentation dynamics in maize columella cells support a new model for the gravity-sensing apparatus of roots

Quantitative analysis of statolith sedimentation behavior was accomplished using videomicroscopy of living columella cells of corn (Zea mays) roots, which displayed no systematic cytoplasmic streaming. Following 90 degrees rotation of the root, the statoliths moved downward along the distal wall and then spread out along the bottom with an average velocity of 1.7 microm min(-1). When statolith trajectories traversed the complete width or length of the cell, they initially moved horizontally toward channel-initiation sites and then moved vertically through the channels to the lower side of the reoriented cell where they again dispersed. These statoliths exhibited a significantly lower average velocity than those sedimenting on distal-to-side trajectories. In addition, although statoliths undergoing distal-to-side sedimentation began at their highest velocity and slowed monotonically as they approached the lower cell membrane, statoliths crossing the cell's central region remained slow initially and accelerated to maximum speed once they reached a channel. The statoliths accelerated sooner, and the channeling effect was less pronounced in roots treated with cytochalasin D. Parallel ultrastructural studies of high-pressure frozen-freeze-substituted columella cells suggest that the low-resistance statolith pathway in the cell periphery corresponds to the sharp interface between the endoplasmic reticulum (ER)-rich cortical and the ER-devoid central region of these cells. The central region is also shown to contain an actin-based cytoskeletal network in which the individual, straight, actin-like filaments are randomly distributed. To explain these findings as well as the results of physical simulation experiments, we have formulated a new, tensegrity-based model of gravity sensing in columella cells. This model envisages the cytoplasm as pervaded by an actin-based cytoskeletal network that is denser in the ER-devoid central region than in the ER-rich cell cortex and is linked to stretch receptors in the plasma membrane. Sedimenting statoliths are postulated to produce a directional signal by locally disrupting the network and thereby altering the balance of forces acting on the receptors in different plasma membrane regions.     

Hydrotropism interacts with gravitropism by degrading amyloplasts in seedling roots of Arabidopsis and radish

In response to a moisture gradient, roots exhibit hydrotropism to control the orientation of their growth. To exhibit hydrotropism, however, they must overcome the gravitropism that is dominant on Earth. We found that moisture gradient or water stress caused immediate degradation of the starch anchors, amyloplasts, in root columella cells of Arabidopsis and radish (Raphanus sativus). Namely, development of hydrotropic response was accompanied by a simultaneous reduction in starch content in columella cells. Rapid degradation of amyloplasts in columella cells also occurred in the water-stressed roots with sorbitol or mannitol. Both hydrotropically stimulated and water-stressed roots showed a reduced responsiveness to gravity. Roots of a starchless mutant, pgm1-1, showed an enhanced hydrotropism compared with that of the wild type. These results suggest that the reduced responsiveness to gravity is, at least in part, attributable to the degradation of amyloplasts in columella cells. Thus, the reduction in gravitropism allows the roots to exhibit hydrotropism.     

A Morphometric Analysis of the Redistribution of Organelles in Columella Cells in Primary Roots of Normal Seedlings and Agravitropic Mutants of Hordeum vulgare

Moore, R. 1985. A morphometric analysis of the redistributionof organellcs in columella cells in primary roots of normalseedlings and agravitropic mutants of Hordeum vulgare.—J.exp. Bot. 36:1275–1286. The redistribution of organeUes m columella cells of horizontally-orientedroots of Hordeum vulgare was quantified in order to determinewhat structural changes in graviperceptive (i.e, columella)cells are associated with the onset of root gravicurvature.The sedimentation of amyloplasts is the only major change incellular structure that correlates positively with the onsetof root gravicurvature, which begins within 15 min after re-orientation.There is no consistent contact between sedimented amyloplastsand any other organelles. Nuclei are restricted to the proximalends of columella cells in vertically-oriented roots, and remainthere throughout gravicurvature after roots are oriented horizontally.Root gravicurvature does not involve significant changes in(1) the volume of columella cells, (2) the relative or absolutevolumes of organelles in columella cells, or (3) the distributionof endoplasmic reticulum (ER). The size, number and sedimentationrates of amyloplasts in columella cells of non-graviresponsiveroots of mutant seedlings are not significantly different fromthose of graviresponsive roots of normal seedlings. Similarly,there is no significant difference in (1) cellular volume, (2)distribution or surface area of ER, (3) patterns or rates oforganelle redistribution in horizontally-oriented roots, or(4) relative or absolute volumes of organelles in columellacells of graviresponsive and non-graviresponsive roots. Theseresults suggest that the lack of gravi-responsiveness by rootsof mutant seedlings is probably not due to either (1) structuraldifferences in columella cells, or (2) differences in patternsor rates of organelle redistribution as compared to that characteristicof graviresponsive roots. Thus, the basis of non-graviresponsivenessin this mutant is probably different from other agravitropicmutants so far studied. Key words: Agravitropic mutant, barley, columella cell, gravitropism (root), Hordeum vulgare, ultrastructure     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

Touch modulates gravity sensing to regulate the growth of primary roots of Arabidopsis thaliana

Plants must sense and respond to diverse stimuli to optimize the architecture of their root system for water and nutrient scavenging and anchorage. We have therefore analyzed how information from two of these stimuli, touch and gravity, are integrated to direct root growth. In Arabidopsis thaliana, touch stimulation provided by a glass barrier placed across the direction of growth caused the root to form a step-like growth habit with bends forming in the central and later the distal elongation zones. This response led to the main root axis growing parallel to, but not touching the obstacle, whilst the root cap maintained contact with the barrier. Removal of the graviperceptive columella cells of the root cap using laser ablation reduced the bending response of the distal elongation zone. Similarly, although the roots of the gravisensing impaired pgm1-1 mutant grew along the barrier at the same average angle as wild-type, this angle became more variable with time. These observations imply a constant gravitropic re-setting of the root tip response to touch stimulation from the barrier. In wild-type plants, transient touch stimulation of root cap cells, but not other regions of the root, inhibited both subsequent gravitropic growth and amyloplast sedimentation in the columella. Taken together, these results suggest that the cells of the root cap sense touch stimuli and their subsequent signaling acts on the columella cells to modulate their graviresponse. This interaction of touch and gravity signaling would then direct root growth to avoid obstacles in the soil while generally maintaining downward growth.     

ROOT APEX STRUCTURE IN EPHEDRA MONOSPERMA AND EPHEDRA CHILENSIS (EPHEDRACEAE)

The root meristem of E. monosperma and E. chilensis possesses a central group of distinctive, large cells. These cells have large nuclei with scattered heterochromatin, proplastids with no starch, small vacuoles, mitochondria, few dictyosomes and endoplasmic reticulum cisternae, and lipid deposits. Over a 24 hr labelling period, the large cells fail to incorporate ³H-thymidine, whereas cells both distal and proximal to this region do. A quiescent center which includes these large cells is present therefore. Both species have an extensive root cap, the length being contributed by mitoses in many tiers of cells distal to the quiescent center. The root cap consists of a columella and peripheral regions. Distinctive amyloplasts, an increase in the number of endoplasmic reticulum cisternae and dictyosomes, large vacuoles, and lipid deposits are characteristic of differentiated columella cells. Peripheral cells elongate, lose most of their starch, and are eventually sloughed from the root.     

Morphogenesis of the root cap in adventitious roots of Salix viminalis

The preformed root primordia in stems of Salk viminalis L. consist of undifferentiated cells. Forty-eight hours after activation of the primordia in cuttings a root cap meristem was initiated four to five cell tiers from the surface of the primordia. The cells distal to the meristem divided only in an anticlinal plane, while in the meristem they divided mostly periclinally but sometimes anticlinally. After 72 hours a columella was established and the amyloplasts began to sediment in response to gravity. Shortly after this stage the roots began to bend slightly downward, probably as a geo-tropic response. Six days after activation the root cap consisted of up to 15 tiers of cells. The ultrastucture of the cap cells just prior to emergence was studied in more detail. The plastids in the cells adjoining the root proper were typical proplastids. Distal to this cell tier starch accumulated in the plastids. In the fifth tier the amyloplasts were fully sedimented to the lowermost cell walls. The amount of ER increased with the distance from the initial cells and most of it was located at the distal periclinal cell wall. The nucleus and the vacuoles in the geo-sensitive cells occurred in the space above the sedimented amyloplasts. The cytoplasm was less electron opaque than in the initial cells and the mitochondria had more cristae. In the distal cells of the columella and the lateral root cap secretion of mucilage seemed to have started. Numerous large dictyosomes were associated with large vesicles containing a fibrillar or granular material. The plasmalemma lining the distal periclinal cell wall had separated from the wall. A fibrillar material was present between the plasmalemma and the wall and also in intercellular spaces outside the root cap.     

Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro(35S):MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.